Tissue grafts modified with a cross-linking agent and method of making and using the same

ABSTRACT

Described herein are tissue grafts derived from the placenta that possess good adhesion to biological tissues and are useful in would healing applications. In one aspect, the tissue graft includes (1) two or more layers of amnion, wherein at least one layer of amnion is cross-linked, (2) two or more layers of chorion, wherein at least one layer of amnion is cross-linked, or (3) one or more layers of amnion and chorion, wherein at least one layer of amnion and/or chorion is cross-linked. In another aspect, the grafts are composed of amnion and chorion cross-linked with one another. In a further aspect, the grafts have one or more layers sandwiched between the amnion and chorion membranes. The amnion and/or the chorion are treated with a cross-linking agent prior to the formation of the graft. Also described herein are methods for making and using the tissue grafts.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority upon U.S. provisional application Ser.No. 61/442,348, filed Feb. 14, 2011. This application is herebyincorporated by reference in its entirety for all of its teachings.

BACKGROUND

The human spinal cord and brain are covered with the meninges membranes,consisting of three overlapping layers of tissue including the outermostdura mater, arachnoid mater and innermost pia mater. The meningealmembranes are critical to the operation of the central nervous systemand their disruption, by accident or surgical intervention, can causeserious consequences unless repaired. Dural tears are commoncomplications in spine surgery and can range in size from nonleakingpinholes to large defects that require tissue reconstruction with apatch. A persistent tear can lead to severe headaches, CSF fistula,formation of a pseudocyst, nerve root entrapment and fluid collection.Large retrospective series have reported an incidence of 1% for cervicalsurgeries (Hannallah D, Lee J, Khan M, Donaldson W F, Kang J D:Cerebrospinal fluid leaks following cervical spine surgery. J Bone JointSurg Am 2008; 90(5):1101-1105), and 7.6% and 15.9%, respectively, forprimary lumbar and revision lumbar surgeries Khan M H, Rihn J, Steele G,et al: Postoperative management protocol for incidental dural tearsduring degenerative lumbar spine surgery: A review of 3,183 consecutivedegenerative lumbar cases. Spine (Phila Pa. 1976)2006:31(22):2609-2613).

A watertight closure is the critical factor in repairing the tear.Direct suture repair is routinely used to repair dural tears, butleakage through suture holes can occur. Autologous fat grafts harvestedthrough the same incision have been used to form a hydrophobic seal toprevent leakage. The graft should be sufficiently large to cover thedura and is sutured to the dura adjacent to the defect (Mayfield F H,Kurokawa K: Watertight closure of spinal dura mater: Technical note. JNeurosurg 1975; 43(5):639-640). Muscle and fascia have also been used aspatches. The use of autologous tissues, however, can lead to prolongedoperating time, blood loss and separate incisions. In addition, thequantity of autologous graft may be inadequate in children. Xenograftsand cross-linked animal-derived collagen matrices can be used as duralpatches, but carry the risk of disease transmission.

Adjunct techniques to assist in sealing the dural tear include fibringlue and hydrogels. Fibrin glue is prepared from pooled blood and hasthe potential to transmit disease. At this time, the application offibrin glue to seal dural tears constitutes off label use. Synthetichydrogels such as the DuraSeal Spine Sealant System (Confluent SurgicalInc., Waltham, Mass.) consist of two components (polyethylene glycolester and trilysine amine) and a delivery system which polymerize at thedefect site to form a seal. As the hydrogel swells to up to 50% in sizeduring polymerization, neural compression may occur.

Post-operative fibrosis, also referred to as scar tissue formation orpost-operative adhesion, is a natural occurrence following varioussurgical procedures. This natural wound healing cascade in mostinstances results in the formation of soft tissue adhesions, whicheither tether, compress, or effect surgical accessibility. Fibrosis isparticularly problematic in post-surgical procedures of the spine. Forexample, peridural fibrosis is the fibroblastic invasion of the nerveroots and peridural sac that are exposed during surgery. The scar tissuecan engulf the dura and nerve roots, which can ultimately result in thereoccurrence of symptoms similar to those experienced prior to surgery.Thus, subsequent operations to address the problem may have to beperformed, which provides additional inconvenience, costs, and risks tothe patient.

Scar formation after spinal surgery can be prevented by the use ofmembranes and foams applied directly to the spine. Currently, membranesused for adhesion prevention are derived from synthetic type I, II, andIII collagen, acellular dermal matrix allograft, HA cellulose film orporcine intestinal submucosa (SIS). These materials possess a variety ofdisadvantages including poor handling characteristics, undesirableresorption profile, limited fixation capabilities, and limited storageoptions. Thus, what is needed are grafts that exhibit good adhesion tobody tissues and facilitate wound healing yet that do not possess thedisadvantages of commercially available options.

SUMMARY

Described herein are tissue grafts derived from the placenta thatpossess good adhesion to biological tissues and are useful in wouldhealing applications. In one aspect, the tissue graft includes (1) twoor more layers of amnion, wherein at least one layer of amnion iscross-linked, (2) two or more layers of chorion, wherein at least onelayer of amnion is cross-linked, or (3) one or more layers of amnion andchorion, wherein at least one layer of amnion and/or chorion iscross-linked. In another aspect, the grafts are composed of amnion andchorion cross-linked with one another. In a further aspect, the graftshave one or more layers sandwiched between the amnion and chorionmembranes. The amnion and/or the chorion are treated with across-linking agent prior to the formation of the graft. The presence ofthe cross-linking agent present on the graft also enhances adhesion tothe biological tissue of interest. Also described herein are methods formaking and using the tissue grafts.

The advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the aspects describedbelow. The advantages described below will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 is an overview flow chart of the process for making the tissuegrafts described herein.

FIG. 2 is a perspective view of an exemplary drying fixture for makingthe tissue grafts described herein.

FIG. 3 is a side view of an amnion/chorion tissue graft describedherein.

FIG. 4 shows an exemplary drying fixture and drying rack useful inpreparing tissues grafts described herein.

DETAILED DESCRIPTION

Before the present articles and methods are disclosed and described, itis to be understood that the aspects described below are not limited tospecific compounds, synthetic methods, or uses as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular aspects only and is notintended to be limiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a cross-linking agent” includes mixtures of two or moresuch agents, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally cleaning step” means thatthe cleaning step may or may not be performed.

The term “subject” as used herein is any vertebrate organism.

The term “amnion” as used herein includes amniotic membrane where theintermediate tissue layer is intact or has been substantially removed.

Titles or subtitles may be used in the specification for the convenienceof a reader, which are not intended to influence the scope of thepresent invention. Additionally, some terms used in this specificationare more specifically defined below.

I. Tissue Grafts and Methods for Making Thereof

Described herein are tissue grafts derived from the placenta thatpossess good adhesion to biological tissues and are useful in wouldhealing applications. In one aspect, the grafts are composed of amnionand/or chorion cross-linked with one another. The amnion and/or thechorion are treated with a cross-linking agent prior to the formation ofthe graft.

In one aspect, the method for making an amnion tissue graft involves:

(a) providing a first amnion having a first surface and a secondsurface; and(b) laminating a second amnion or chorion having a first surface and asecond surface to the second surface of the first amnion, where thefirst surface of the second amnion or chorion is laminated to the secondsurface of the first amnion, and wherein the first amnion, secondamnion, and/or the chorion are treated with a cross-linking agent priorto lamination.

In another aspect, the method for making a chorion or chorion/amniontissue graft involves:

(a) providing a first chorion having a first surface and a secondsurface; and(b) laminating a second chorion having a first surface and a secondsurface to the second surface of the first chorion, where the firstsurface of the second chorion is laminated to the second surface of thefirst chorion, and wherein the first chorion and/or second chorion aretreated with a cross-linking agent prior to lamination.

In another aspect, the method for making a the tissue graft involves:

(a) optionally removing substantially all of the epithelium cells of theamnion to expose the basement membrane of the amnion to produce a firstmembrane having an expose basement membrane and a second surface;(b) treating the first membrane with a first cross-linking agent; and(c) mounting a chorion having a stromal layer and a second surface tothe amnion, wherein the stromal layer of the chorion is adjacent to thesecond surface of the amnion, wherein the chorion is treated with asecond cross-linking agent, and wherein the first cross-linking agentand the second cross-linking agent are the same or different.

In another aspect, the tissue graft is produced by the method involving:

(a) providing a first amnion having a first surface and a secondsurface; and(b) laminating a second amnion or first chorion having a first surfaceand a second surface to the second surface of the first amnion, wherethe first surface of the second amnion or first chorion is laminated tothe second surface of the first amnion to produce a laminated article,and(c) contacting the laminated article with a cross-linking agent.

In another aspect, the tissue graft is produced by the method involving:

(a) providing a first chorion having a first surface and a secondsurface; and(b) laminating a second chorion or first amnion having a first surfaceand a second surface to the second surface of the first chorion, wherethe first surface of the second chorion or first amnion is laminated tothe second surface of the first chorion to produce a laminated article,and(c) contacting the laminated article with a cross-linking agent.

In a further aspect, the tissue graft is produced by the methodinvolving:

(a) obtaining a placenta from a subject, wherein the placenta comprisesan amnion and a chorion;(b) cleaning the placenta;(c) separating the chorion tissue layer from the amnion layer, whereinthe amnion comprises epithelium cells adjacent to a basement membrane;(d) optionally removing substantially all of the epithelium cells toexpose the basement membrane of the amnion to produce a first membrane:(e) treating the first membrane and chorion with one or morecross-linking agents;(f) mounting the first membrane onto a surface of a drying fixture,wherein the basement membrane of the first membrane is adjacent to thesurface of the drying fixture;(g) mounting a chorion on the first membrane to produce the tissuegraft; and(h) dehydrating the tissue graft on the drying fixture.

FIG. 1 depicts an exemplary overview (100) and certain aspects of thesteps to harvest, process, and prepare placental material for later useas a tissue graft. More detailed descriptions and discussion regardingeach individual step will follow. Initially, the placenta tissue iscollected from a consenting patient following an elective Cesareansurgery (step 110). The material is preserved and transported inconventional tissue preservation manner to a suitable processinglocation or facility for check-in and evaluation (step 120). Grossprocessing, handling, and separation of the amnion and chorion thentakes place (step 130). Acceptable tissue is then decontaminated (step140), followed by the optional step of substantially removing theepithelium layer from the amnion to expose the basement membrane (step145). Next, the amnion and/or chorion are treated with a cross-linkingagent solution (step 147). The tissue graft is then prepared from theamnion and/or chorion and the graft is subsequently dehydrated (step150), cut and packaged (step 160), sterilized using gamma radiation orelectron beam radiation (step 165), and released (step 170) to themarket for use by surgeons and other medical professionals inappropriate surgical procedures and for wound care. Each step isdescribed in detail below.

Initial Tissue Collection (Step 110)

The components used to produce the tissue grafts are derived from theplacenta. The source of the placenta can vary. In one aspect, theplacenta is derived from a mammal such as human and other animalsincluding, but not limited to, cows, pigs, and the like can be usedherein. In the case of humans, the recovery of the placenta originatesin a hospital, where it is collected during a Cesarean section birth.The donor, referring to the mother who is about to give birth,voluntarily submits to a comprehensive screening process designed toprovide the safest tissue possible for transplantation. The screeningprocess preferably tests for antibodies to the human immunodeficiencyvirus type 1 and type 2 (anti-HIV-1 and anti-HIV-2), antibodies to thehepatitis B virus (anti-HBV) hepatitis B surface antigens (HBsAg),antibodies to the hepatitis C virus (anti-HCV), antibodies to the humanT-lymphotropic virus type I and type II (anti-HTLV-I, anti-HTLV-II),CMV, and syphilis, and nucleic acid testing for human immune-deficiencyvirus type 1 (HIV-1) and for the hepatitis C virus (HCV), usingconventional serological tests. The above list of tests is exemplaryonly, as more, fewer, or different tests may be desired or necessaryover time or based upon the intended use of the grafts, as will beappreciated by those skilled in the art.

Based upon a review of the donor's information and screening testresults, the donor will either be deemed acceptable or not. In addition,at the time of delivery, cultures are taken to determine the presence ofbacteria, for example, Clostridium or Streptococcus. If the donor'sinformation, screening tests, and the delivery cultures are allsatisfactory (i.e., do not indicate any risks or indicate acceptablelevel of risk), the donor is approved by a medical director and thetissue specimen is designated as initially eligible for furtherprocessing and evaluation.

Human placentas that meet the above selection criteria are preferablybagged in a saline solution in a sterile shipment bag and stored in acontainer of wet ice for shipment to a processing location or laboratoryfor further processing.

If the placenta is collected prior to the completion of obtaining theresults from the screening tests and delivery cultures, such tissue islabeled and kept in quarantine. The placenta is approved for furtherprocessing only after the required screening assessments and deliverycultures, which declare the tissue safe for handling and use, aresatisfied and obtains final approval from a medical director.

Material Check-in and Evaluation (Step 120)

Upon arrival at the processing center or laboratory, the shipment isopened and verified that the sterile shipment bag/container is stillsealed and in the coolant, that the appropriate donor paperwork ispresent, and that the donor number on the paperwork matches the numberon the sterile shipment bag containing the tissue. The sterile shipmentbag containing the tissue is then stored in a refrigerator until readyfor further processing.

Gross Tissue Processing (Step 130)

When the tissue is ready to be processed further, the sterile suppliesnecessary for processing the placental tissue further are assembled in astaging area in a controlled environment and are prepared forintroduction into a controlled environment. In one aspect, the placentais processed at room temperature. If the controlled environment is amanufacturing hood, the sterile supplies are opened and placed into thehood using conventional sterilization techniques. If the controlledenvironment is a clean room, the sterile supplies are opened and placedon a cart covered by a sterile drape. All the work surfaces are coveredby a piece of sterile drape using conventional sterilization techniques,and the sterile supplies and the processing equipment are placed ontothe sterile drape, again using conventional sterilization techniques.

Processing equipment is decontaminated according to conventional andindustry-approved decontamination procedures and then introduced intothe controlled environment. The equipment is strategically placed withinthe controlled environment to minimize the chance for the equipment tocome in proximity to or is inadvertently contaminated by the tissuespecimen.

Next, the placenta is removed from the sterile shipment bag andtransferred aseptically to a sterile processing basin within thecontrolled environment. The sterile basin contains hyperisotonic salinesolution (e.g., 18% NaCl) that is at room or near room temperature. Theplacenta is gently massaged to help separate blood clots and to allowthe placental tissue to reach room temperature, which facilitates theseparation of the placental components from each other (e.g., amnionmembrane and chorion). After having warmed up to ambient temperature(e.g., after about 10-30 minutes), the placenta is then removed from thesterile processing basin and laid flat on a processing tray with theamnion membrane layer facing down for inspection.

The placenta is examined for discoloration, debris or othercontamination, odor, and signs of damage. The size of the tissue is alsonoted. A determination is made, at this point, as to whether the tissueis acceptable for further processing.

The amnion and chorion are next carefully separated. In one aspect, thematerials and equipment used in this procedure include a processingtray, 18% saline solution, sterile 4×4 sponges, and two sterile Nalgenejars. The placenta tissue is then closely examined to find an area(typically a corner) in which the amnion can be separated from thechorion. The amnion appears as a thin, opaque layer on the chorion.

The fibroblast layer is identified by gently contacting each side of theamnion with a piece of sterile gauze or a cotton tipped applicator. Thefibroblast layer will stick to the test material. The amnion is placedinto processing tray basement membrane layer down. Using a bluntinstrument, a cell scraper, or sterile gauze, any residual blood is alsoremoved. This step must be done with adequate care, again, so as not totear the amnion. The cleaning of the amnion is complete once the amnionis smooth and opaque-white in appearance.

In certain aspects, the intermediate tissue layer, also referred to asthe spongy layer, is substantially removed from the amnion in order toexpose the fibroblast layer. The term “substantially removed” withrespect to the amount of intermediate tissue layer removed is definedherein as removing greater than 90%, greater than 95%, or greater than99% of the intermediate tissue layer from the amnion. This can beperformed by peeling the intermediate tissue layer from the amnion.Alternatively, the intermediate tissue layer can be removed from theamnion by wiping the intermediate tissue layer with gauze or othersuitable wipe. The resulting amnion can be subsequently decontaminatedusing the process described below. Not wishing to be bound by theory,the removal of the intermediate layer can accelerate the drying of thetissue graft, particularly if multiple amnion membranes are used toproduce the graft. The intermediate layer can be removed from the amnionprior contacting the amnion with the cross-linking agent or, in thealternative, can be removed after the amnion has been contacted with thecross-linking agent.

Chemical Decontamination (Step 140)

The amnion and chorion isolated above can be chemically decontaminatedusing the techniques described below. In one aspect, the amnion andchorion is decontaminated at room temperature. In one aspect, the amnionproduced in step 130 can be placed into a sterile Nalgene jar for thenext step. In one aspect, the following procedure can be used to cleanthe amnion. A Nalgene jar is aseptically filled with 18% salinehypertonic solution and sealed (or sealed with a top). The jar is thenplaced on a rocker platform and agitated for between 30 and 90 minutes,which further cleans the amnion of contaminants. If the rocker platformwas not in the critical environment (e.g., the manufacturing hood), theNalgene jar is returned to the controlled/sterile environment andopened. Using sterile forceps or by aseptically decanting the contents,the amnion is gently removed from the Nalgene jar containing the 18%hyperisotonic saline solution and placed into an empty Nalgene jar. Thisempty Nalgene jar with the amnion is then aseptically filled with apre-mixed antibiotic solution. In one aspect, the premixed antibioticsolution is composed of a cocktail of antibiotics, such as StreptomycinSulfate and Gentamicin Sulfate. Other antibiotics, such as Polymixin BSulfate and Bacitracin, or similar antibiotics now available oravailable in the future, are also suitable. Additionally, it ispreferred that the antibiotic solution be at room temperature when addedso that it does not change the temperature of or otherwise damage theamnion. This jar or container containing the amnion and antibiotics isthen sealed or closed and placed on a rocker platform and agitated for,preferably, between 60 and 90 minutes. Such rocking or agitation of theamnion within the antibiotic solution further cleans the tissue ofcontaminants and bacteria. Optionally, the amnion can be washed with adetergent. In one aspect, the amnion can be washed with 0.1 to 10%, 0.1to 5%, 0.1 to 1%, or 0.5% Triton-X wash solution.

If the rocker platform was not in the critical environment (e.g., themanufacturing hood), the jar or container containing the amnion andantibiotics is then returned to the critical/sterile environment andopened. Using sterile forceps, the amnion is gently removed from the jaror container and placed in a sterile basin containing sterile water ornormal saline (0.9% saline solution). The amnion is allowed to soak inplace in the sterile water/normal saline solution for at least 10 to 15minutes. The amnion may be slightly agitated to facilitate removal ofthe antibiotic solution and any other contaminants from the tissue.After at least 10 to 15 minutes, the amnion is ready to be dehydratedand processed further.

In the case of chorion, the following exemplary procedure can be used.After separation of the chorion from the amnion and removal of clottedblood from the fibrous layer, the chorion is rinsed in 18% salinesolution for 15 minutes to 60 minutes. During the first rinse cycle, 18%saline is heated in a sterile container using a laboratory heating platesuch that the solution temperature is approximately 48° C. The solutionis decanted, the chorion tissue is placed into the sterile container,and decanted saline solution is poured into the container. The containeris sealed and placed on a rocker plate and agitated for 15 minutes to 60minutes. After 1 hour agitation bath, the chorion tissue was removed andplaced into second heated agitation bath for an additional 15 minutes to60 minutes rinse cycle. Optionally, the chorion tissue can be washedwith a detergent (e.g., Triton-X wash solution) as discussed above forthe decontamination of amnion. The container is sealed and agitatedwithout heat for 15 minutes to 120 minutes. The chorion tissue is nextwashed with deionized water (250 ml of DI water×4) with vigorous motionfor each rinse. The tissue is removed and placed into a container of1×PBS w/EDTA solution. The container is sealed and agitated for 1 hourat controlled temperature for 8 hours. The chorion tissue is removed andrinsed using sterile water. A visual inspection was performed to removeany remaining discolored fibrous blood material from the chorion tissue.The chorion tissue should have a cream white visual appearance with noevidence of brownish discoloration.

Optional Removal of Epithelium Layer from Amnion (Step 145)

In certain aspects, it is desirable to remove the epithelium layerpresent on the amnion. In one aspect, the epithelium layer present onthe amnion is substantially removed in order to expose the basementlayer of the amnion. The term “substantially removed” with respect tothe amount of epithelium removed is defined herein as removing greaterthan 90%, greater than 95%, or greater than 99% of the epithelial cellsfrom the amnion. The presence or absence of epithelial cells remainingon the amnion layer can be evaluated using techniques known in the art.For example, after removal of the epithelial cell layer, arepresentative tissue sample from the processing lot is placed onto astandard microscope examination slide. The tissue sample is then stainedusing Eosin Y Stain and evaluated as described below. The sample is thencovered and allowed to stand. Once an adequate amount of time has passedto allow for staining, visual observation is done under magnification.

The epithelium layer can be removed by techniques known in the art. Forexample, the epithelium layer can be scraped off of the amnion using acell scraper. Other techniques include, but are not limited to, freezingthe membrane, physical removal using a cell scraper, or exposing theepithelial cells to nonionic detergents, anionic detergents, andnucleases. The de-epithelialized tissue is then evaluated to determinethat the basement membrane has not been compromised and remains intact.This step is performed after completion of the processing step and thebefore the tissue has been dehydrated as described in the next section.For example, a representative sample graft is removed for microscopicanalysis. The tissue sample is place onto a standard slide, stained withEosin Y and viewed under the microscope. If epithelium is present, itwill appear as cobblestone-shaped cells.

The methods described herein, particularly steps 130 and 145, do notremove all cellular components in the amnion. This technique is referredto in the art as “decellularization.” Decellularization generallyinvolves the physical and/or chemical removal of all cells present inthe amnion, which includes epithelial cells and fibroblast cells.Although step 145 does remove epithelial cells, the fibroblast layerpresent in the amnion stromal layer is intact, even after removal of theintermediate layer discussed in step 130.

Treatment with a Cross-Linking Agent (Step 147)

Depending upon the application of the tissue graft, the amnion and/orchorion are individually treated with a cross-linking agent prior tolamination. In another aspect, a laminate composed of (1) two or morelayers of amnion, (2) two or more layers of chorion, or (3) one or morelayers of amnion and chorion can subsequently be treated with across-linking agent after lamination.

In general, the cross-linking agent is nontoxic and non-immunogenic.When the chorion and amnion are treated with the cross-linking agent,the cross-linking agent can be the same or different. In one aspect, thechorion and amnion can be treated separately with a cross-linking agentor, in the alternative, the chorion and amnion can be treated togetherwith the same cross-linking agent. In certain aspects, the amnion orchorion can be treated with two or more different cross-linking agents.The conditions for treating the amnion and/or chorion can vary. In oneaspect, the amnion or chorion can be placed in a container holding anaqueous solution of the cross-linking agent. In one aspect, theconcentration of the cross-linking agent is from 0.1 M to 5 M, 0.1 M to4 M, 0.1 M to 3 M, 0.1 M to 2 M, or 0.1 M to 1 M. In another aspect, theamnion or chorion are treated with the cross-linking agent for 1 to 2seconds up to 60 minutes. In a further aspect, the amnion or chorion aretreated with the cross-linking agent at room temperature up to 50° C.

The cross-linking agent generally possesses two or more functionalgroups capable of reacting with proteins to produce covalent bonds. Inone aspect, the cross-linking agent possesses groups that can react withamino groups present on the protein. Examples of such functional groupsinclude, but are not limited to, hydroxyl groups, substituted orunsubstituted amino groups, carboxyl groups, and aldehyde groups. In oneaspect, the cross-linker can be a dialdehydes such as, for example,glutaraldehyde. In another aspect, the cross-linker can be acarbodiimide such as, for example,(N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide (EDC). In otheraspects, the cross-linker can be an oxidized dextran, p-azidobenzoylhydrazide, N-[alpha-maleimidoacetoxy]succinimide ester, p-azidophenylglyoxal monohydrate, bis-[beta-(4-azidosalicylamido)ethyl]disulfide,bis-[sulfosuccinimidyl]suberate, dithiobis[succinimidyl]propionate,disuccinimidyl suberate, and1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, abifunctional oxirane (OXR), or ethylene glycol diglycidyl ether (EGDE).

In one aspect, sugar is the cross-linking agent, where the sugar canreact with proteins present in the amnion and chorion to form a covalentbond. For example, the sugar can react with proteins by the Maillardreaction, which is initiated by the nonenzymatic glycosylation of aminogroups on proteins by reducing sugars and leads to the subsequentformation of covalent bonds. Examples of sugars useful as across-linking agent include, but are not limited to, D-ribose,glycerose, altrose, talose, ertheose, glucose, lyxose, mannose, xylose,gulose, arabinose, idose, allose, galactose, maltose, lactose, sucrose,cellibiose, gentibiose, melibiose, turanose, trehalose, isomaltose, orany combination thereof. Thus, in one aspect, the amnion or chorioninclude at least one cross-linker covalently attached to the membrane.In another aspect, a tissue graft includes an amnion and a chorionlaminate, wherein the amnion and chorion are covalently attached to oneanother via a cross-linker.

The following procedure provides an exemplary method for treating theamnion and chorion with a cross-linking agent. The cleaned anddecontaminated chorion and amnion are placed on the sterile field in themanufacturing hood. The tissue is transferred to a Nalgene jarcontaining a cross-linking agent, preferably 0.05 to 1 M D-ribose,preferably 0.2 M (3.01%) D-ribose, for 1 to 60 minutes, preferably 5minutes. The tissues may be treated with the cross-linking agent eitherin separate containers or together in the same container. After theincubation, the tissue is removed from the solution and, optionally,allowed to dry.

Preparation of Tissue Graft and Dehydration (Step 150)

After the amnion and/or chorion have been treated with the cross-linkingagent individually or as a laminate as discussed in the previoussection, a tissue graft composed of the amnion and/or chorion isproduced and subsequently dehydrated. In one aspect, the tissue graft isa laminate composed of two or more amnion, two or more chorion, oramnion and chorion layered on top of each other. In one aspect, thetissue graft is dehydrated by chemical dehydration followed byfreeze-drying. In one aspect, the chemical dehydration step is performedby contacting the amnion and chorion independently or as a laminate witha polar organic solvent for a sufficient time and amount in order tosubstantially (i.e., greater than 90%, greater than 95%, or greater than99%) or completely remove residual water present in the amnion orchorion (i.e., dehydrate the tissue). The solvent can be protic oraprotic. Examples of polar organic solvents useful herein include, butare not limited to, alcohols, ketones, ethers, aldehydes, or anycombination thereof. Specific, non-limiting examples include DMSO,acetone, tetrahydrofuran, ethanol, isopropanol, or any combinationthereof. In one aspect, the placental tissue is contacted with a polarorganic solvent at room temperature. No additional steps are required,and the tissue can be freeze-dried directly as discussed below.

After chemical dehydration, the tissue graft is freeze-dried in order toremove any residual water and polar organic solvent. In one aspect, theamnion and chorion can be laid on a suitable drying fixture prior tofreeze-drying. In one aspect, amnion treated with a cross-linking agentis laid on a suitable drying fixture, where the exposed basement (i.e.,epithelium layer substantially removed) or unexposed basement membrane(i.e., epithelium layer not removed) is adjacent to the surface of thedrying fixture.

In one aspect, additional amnion membrane(s) (cross-linkingagent-treated or untreated) can be applied to the first amnion affixedto the drying fixture prior to application of the chorion (treated oruntreated with cross-linking agent). In these aspects, it is notnecessary to remove the epithelial cells from the basement membrane forthose layers that are not in direct contact with host cells. Although inthis aspect, the fibroblast layer is used to adhere the membranestogether, other techniques and materials such as, for example, fibringlue, gelatin, photochemical techniques, and suturing can be used toproduce the multi-laminated tissue graft. The actual number of layerswill depend upon the surgical need and procedure with which the tissuegraft is designed to be used for. In general, the tissue graftsdescribed herein are cut to size to match the morphology of the wound,placed on or within the wound, and if desired, can be held in place withsutures or surgical adhesives to augment the cross-linking agent.

In other aspects, the amnion can be placed on the surface of the dryingfixture such that the exposed basement or unexposed basement membrane isfacing up. In certain aspects, one or more additional membranes can belaminated between the amnion and chorion membranes. In this aspect, theadditional membrane(s) can optionally be treated with a cross-linkingagent. Examples of additional membranes include, but are not limited to,allograft pericardium, allograft acelluar dermis, amnion, chorion,Wharton's jelly, purified xenograft Type-1 collagen, biocellulosepolymers or copolymers, biocompatible synthetic polymer or copolymerfilms, purified small intestinal submucosa, bladder acellular matrix,cadaveric fasia, or any combination thereof, wherein any of themembranes can optionally be treated with a cross-linking agent.

The drying fixture is preferably sized to be large enough to receive theamnion and chorion, fully, in laid out, flat fashion. In one aspect, thedrying fixture is made of Teflon or of Delrin, which is the brand namefor an acetal resin engineering plastic invented and sold by DuPont andwhich is also available commercially from Werner Machine, Inc. inMarietta, Ga. Any other suitable material that is heat and cutresistant, capable of being formed into an appropriate shape to receivewet tissue can also be used for the drying fixture.

In one aspect, similar to that shown in FIG. 2, the receiving surface ofthe drying fixture 500 has grooves 505 that define the product spaces510, which are the desired outer contours of the tissue after it is cutand of a size and shape that is desired for the applicable surgicalprocedure in which the tissue will be used. For example, the dryingfixture can be laid out so that the grooves are in a grid arrangement.The grids on a single drying fixture may be the same uniform size or mayinclude multiple sizes that are designed for different surgicalapplications. Nevertheless, any size and shape arrangement can be usedfor the drying fixture, as will be appreciated by those skilled in theart. In another embodiment, instead of having grooves to define theproduct spaces, the drying fixture has raised ridges or blades.

Within the “empty” space between the grooves or ridges, the dryingfixture can include a slightly raised or indented texture in the form oftext, logo, name, or similar design 520. This textured text, logo, name,or design can be customized. When dried, the tissue will mold itselfaround the raised texture or into the indented texture—essentiallyproviding a label within the tissue itself. Preferably, thetexture/label can be read or viewed on the tissue graft in only oneorientation so that, after drying and cutting, an end user (typically, aclinician) of the dried tissue will be able to tell the stromal sidefrom the basement side of the dried tissue. The reason this is desiredis because, during a surgical procedure, it is desirable to place theallograft in place, with amnion basement side down or adjacent thenative tissue of the patient receiving the allograft. FIG. 2 illustratesa variety of marks, logos, and text 520 that can be included within theempty spaces 510 of the drying fixture 500. Typically, a single dryingfixture will include the same design or text within all of the emptyspaces; however, FIG. 2 shows, for illustrative purposes, a wide varietyof designs that can be included on such drying fixtures to emboss eachgraft.

Once the tissue graft composed of amnion and/or chorion is placed on thedrying fixture, the drying fixture is placed in the freeze-dryer. Theuse of the freeze-dryer to dehydrate the tissue grafts can be moreefficient and thorough compared to other techniques such as thermaldehydration. In general, it is desirable to avoid ice crystal formationin the placental tissue grafts as this may damage the extracellularmatrix in the tissue graft. By chemically dehydrating the amnion andchorion prior to freeze-drying, this problem can be avoided.

In another aspect, the dehydration step involves applying heat to thetissue graft. In one aspect, the amnion and/or chorion is laid on asuitable drying fixture as discussed above, and the drying fixture isplaced in a sterile Tyvex (or similar, breathable, heat-resistant, andsealable material) dehydration bag and sealed. The breathabledehydration bag prevents the tissue from drying too quickly. If multipledrying fixtures are being processed simultaneously, each drying fixtureis either placed in its own Tyvex bag or, alternatively, placed into asuitable mounting frame that is designed to hold multiple drying framesthereon and the entire frame is then placed into a larger, singlesterile Tyvex dehydration bag and sealed.

The Tyvex dehydration bag containing the one or more drying fixtures isthen placed into a non-vacuum oven or incubator that has been preheatedto approximately 35 to 50 degrees Celcius. The Tyvex bag remains in theoven for between 30 to 120 minutes. In one aspect, the heating step canbe performed at 45 minutes at a temperature of approximately 45 degreesCelcius to dry the tissue sufficiently but without over-drying orburning the tissue graft. The specific temperature and time for anyspecific oven will need to be calibrated and adjusted based on otherfactors including altitude, size of the oven, accuracy of the oventemperature, material used for the drying fixture, number of dryingfixtures being dried simultaneously, whether a single or multiple framesof drying fixtures are dried simultaneously, and the like.

In one aspect, after the layer of amnion with the exposed basement(i.e., epithelium layer substantially removed) or unexposed basementmembrane (i.e., epithelium layer not removed) has been applied to thedrying fixture, the chorion (untreated or treated with a cross-linkingagent) is applied to the amnion. In one aspect, the exposed basementlayer or unexposed basement layer of the amnion is applied to the dryingfixture, and the chorion membrane (treated or untreated withcross-linking agent) is subsequently applied to the amnion affixed tothe drying fixture. This aspect is depicted in FIG. 3, where the amnionlayer 800 is applied to drying fixture 600, and chorion layer 810 isapplied to the amnion layer 800. In other aspects, once the amnion (andother membranes described below) and chorion have been applied to thedrying fixture, a drying frame can be applied over membranes. Thisfeature is depicted in FIG. 4, where the drying rack 82 is placed on topof drying fixture 80. The drying frame holds the membranes in place.Additionally, the drying frame allows the entire sheet of tissue graftto dry completely without lifting, which results in increased yields.

In certain aspects the tissue graft is not physically altered except forfinal cutting and packaging (step 160). When completed, the processedtissue graft has a semi-transparent appearance with a whitishcoloration. The tissue graft is pliable to withstand bending and sizingin its dry, non-hydrated state. The tissue grafts described herein canbe stored at room temperature for extended periods of time.

Cutting & Packaging (Step 160)

Once the graft has been adequately dehydrated, the tissue graft is thenready to be cut into specific product sizes and appropriately packagedfor storage, terminal sterilization, and later surgical use. In oneaspect, the Tyvek bag containing the dehydrated tissue is placed backinto the sterile/controlled environment. The number of grafts to beproduced is estimated based on the size and shape of the tissue on thedrying fixture(s). An appropriate number of pouches, one for each tissuegraft, is also introduced into the sterile/controlled environment. Thedrying fixture(s) are then removed from the Tyvek bag.

If the drying fixture has grooves, then the following exemplaryprocedure can be used for cutting the tissue graft into product sizes.If the drying fixture is configured in a grid pattern, a #20 or similarstraight or rolling blade is used to cut along each groove line inparallel. Next, all lines in the perpendicular direction are cut.Alternatively, if the drying fixture has raised edges or blades, thenthe following procedure can be used for cutting the tissue graft intoproduct sizes. A sterile roller is used to roll across the dryingfixture. Sufficient pressure must be applied so that the dehydratedtissue graft is cut along all of the raised blades or edges of thedrying fixture.

After cutting, each tissue graft is placed in a respective “inner”pouch. The inner pouch, which preferably has a clear side and an opaqueside, should be oriented clear side facing up. The tissue graft isplaced in the “inner” pouch so that the texture in the form of text,logo, name, or similar design is facing out through the clear side ofthe inner pouch and is visible outside of the inner pouch. This processis repeated for each separate tissue graft.

Each tissue graft is then given a final inspection to confirm that thereare no tears or holes, that the product size (as cut) is withinapproximately 1 millimeter (plus or minus) of the specified length andwidth size and within approximately 250 microns (plus or minus) thickfor that particular graft, that there are no noticeable blemishes ordiscoloration of the tissue graft, and that the textured logo or wordingis readable and viewable through the “inner” pouch.

To the extent possible, oxygen is removed from the inner pouch before itis sealed. The inner pouch can be sealed in any suitable manner;however, a heat seal has shown to be effective. In one aspect, afterpackaging, the product is terminally sterilized by radiation, usinggamma or electron beam sterilization with a target dose of, for example,17.5 kGy. Next, each inner pouch is separately packaged in an “outer”pouch for further protection, storage, and shipment.

It should be noted that none of the steps described above involvefreezing the tissue graft to kill unwanted cells, to decontaminate thetissue graft, or otherwise to preserve the tissue graft. The dehydratedtissue grafts described herein are designed to be stored and shipped atroom or ambient temperature without need for refrigeration or freezing.

Product Release (Step 170)

Before the tissue graft is ready for shipment and release to the enduser, all documentation related to the manufacture, recovery and donoreligibility are reviewed and deemed acceptable by the quality assurancedepartment and the medical director.

Appropriate labeling and chain of custody is observed throughout all ofthe above processes, in accordance with accepted industry standards andpractice. Appropriate clean room and sterile working conditions aremaintained and used, to the extent possible, throughout the aboveprocesses.

II. Applications of Tissue Grafts

Due to the enhanced adhesive nature of the tissue grafts describedherein, the grafts can be used in numerous medical applicationsinvolving wound healing in a subject. Not wishing to be bound by theory,the cross-linking groups covalently attached to the tissue graft canfacilitate the non-enzymatic cross-linking of proteins within the graftsuch as, for example, collagen, and other proteins present in abiological tissue. In one aspect, tissue grafts described herein cancross-link (i.e., form a covalent bond) with dura matter. In otheraspects, the tissue grafts described herein can adhere to tendons,ligaments, muscle, and other body tissue. The tissue grafts describedherein are useful in the reinforcement and sealing of tears as well asthe prevention or reduction of scar formation after surgery in additionto other post-surgical complications. Additionally, due to the enhancedadhesive properties of the tissue graft, the grafts are ready forapplication to the surgical site without the need for sutures.

In one aspect, the grafts described herein are useful in enhancing orimproving wound healing. The types of wounds that present themselves tophysicians on a daily bases are diverse. Acute wounds are caused bysurgical intervention, trauma and burns. Chronic wounds are wounds thatare delayed in closing compared to healing in an otherwise healthyindividual. Examples of chronic wound types plaguing patients includediabetic foot ulcers, venous leg ulcers, pressure ulcers, arterialulcers, and surgical wounds that become infected.

The physician's goal when treating traumatic wounds is to heal the woundwhile allowing the patient to retain natural function in the area of thewound with minimal scaring and infection. If a wound becomes infected,it can lead to a loss of limb or life. For the most part, physiciansheal these patients without incident. However, physicians dealing withchronic wounds are mainly concerned with closing the wound as quickly aspossible to minimize the risk of an infection that could lead to loss oflimb or life. Chronic wounds are wounds on patients that havecomorbidities that complicate or delay the healing cascade. In oneaspect, the grafts described herein can function as a tissueregeneration template that delivers essential wound healing factors,extracellular matrix proteins and inflammatory mediators to help reduceinflammation, enhance healing, and reduces scar tissue formation.

In another aspect, the tissue grafts described herein are useful foraddressing or alleviating complications to the spine and surroundingregions that occur after surgery. Acute and chronic spinal injuries andpain can be attributed to trauma and/or degenerative changes in thespinal column. For the degenerative patient, there is usually aprogression of possible surgeries depending on the patient's symptomsand disease state. The first surgical option when conservative therapyhas failed is a laminectomy or micro-discectomy. These minimallyinvasive procedures are intended to relieve the pain generator orstenosis of the spinal canal. If there is progression of the disease,then other surgeries may be necessary including, but not limited to, aspinal fusion. Spinal fusions may be achieved through severalapproaches: anterior (from the front through the abdomen), posterior(from the back), or lateral (through the side). Each approach hasadvantages and disadvantages. The goal is typically to remove the spinaldisc, restore disc height and fuse the two spinal vertebrae together tolimit motion and further degradation. There are also surgical optionsfor the surgeon and patient to replace the spinal disc with anartificial disc. Spine trauma is typically treated by fusing the spinelevels or if a vertebrae is crushed, the surgeon may choose to do acorpectomy and fusing across the levels that were affected.

In one aspect, the tissue grafts described herein are useful inpreventing or reducing scar formation on the spine or near the spine andsealing dural tears. Scar formation at or near the spine after surgerycan be very debilitating and possibly require subsequent operations toaddress the symptoms as discussed above. The term “anti-adhesion” isalso used in the art to refer to the prevention of scar tissue at ornear the spine. In other aspects, the tissue grafts described herein canbe used as a protective barrier, where the graft protects the spinaldura from post-surgical trauma from the surrounding surgical site. Forexample, the grafts can prevent damage to the spinal dura caused bysharp edges from newly cut bone such as vertebrae. In other aspects, thetissue grafts can be used for anterior lumbar interbody fusion,posterior lumbar interbody fusion trans-lumbar interbody fusion,anterior cervical discectomy and fusion, micro discectomy, spinal durarepair, and as a dura sealant to prevent CSF leakage.

Depending upon the surgical procedure, the tissue graft can be applieddirectly to the spinal dura, the surrounding region of the spine toinclude nerve roots, or a combination thereof. Due to the uniquestructure of vertebrae, the tissue graft can be cut into any shape ordimension so that it can be placed and affixed at the appropriateposition in the subject. For example, when the tissue graft is used forbi-lateral coverage, membranes in the shape of a rectangle allow thetissue graft to fit around the posterior spinal process, which minimizeslateral movement. In addition to minimizing lateral movement, the tissuegraft can also provide proximal and distal barrier coverage where thespinal lamina has been removed for exposure to the affected area. In oneaspect, to ensure proper placement, the graft can be embossed on theexposed basement membrane of the graft to ensure proper placement of thegraft in the subject. In particular, proper graft placement will ensurethat the basement membrane of the graft is in direct contact with thespinal dura or surrounding region. For example, proper membraneplacement and orientation is important when applying the material inspinal applications where a posterior or anterior approach is utilized.

The grafts are useful in preventing or reducing scar formation that canresult from a variety of surgical procedures associated with the spine.The grafts can be used after any procedure in the neck, mid-back, orlower back. Depending upon the application, the epithelium of the amnionmembrane can be substantially removed. For example, in posteriorprocedures such as a laminectomy or discectomy, the epithelium layer issubstantially removed. Removal of the epithelial cell layer exposes theamnion's basement membrane layer, which increases cell signalingcharacteristics. This up regulation response enhances cellular migrationand expression of anti-inflammatory proteins, which inhibits fibrosis.The spinal dura is typically left unprotected following posteriorprocedures. Thus, the grafts described herein provide an unmet need inthese procedures.

In other aspects, the epithelial cell layer is not removed. For example,in anterior procedures or modified anterior procedures such as AnteriorLumbar Interbody Fusion (ALIF) and Transforaminal Interbody Fusion(TLIF), the amnion epithelium layer is not removed and remains intact.In these aspects, the grafts provide additional protection to thevertebral surgical site by maintaining separation from the peritoneum,larger vessels, and abdominal musculature. The membrane serves as areduced friction anatomical barrier against adhesions and scaring. Forexample, the grafts can prevent scar tissue binding major blood vesselsto the spine. This is a common problem with post-spinal surgery, whichrequires a second surgical procedure to address this.

In another aspect, the tissue grafts are useful in dental applications.For example, the grafts can be used around dental implants or in thetreatment of advanced gingival recession defect. In another aspect, thegrafts can be used in guided tissue regeneration.

In other aspects, the grafts described herein can be used in orthopedicapplications (i.e., sports medicine). Sports medicine includes therepair and reconstruction of various soft-tissue injuries in or aroundjoints caused by traumas, or chronic conditions brought about byrepeated motion, in active individuals and athletes. For example, sportsmedicine includes the treatment of a variety of different injuriesassociated with, but not limited to, shoulders, elbows, feet, ankleshand and wrists.

The main types of injuries include tendon and ligament sprains andruptures in the various joints, with the most common being ACL in theknee and rotator cuff in the shoulder. Non-tendon and ligamentprocedures include repair of torn knee meniscus and repair of kneecartilage which if left un-treated can lead to osteoarthritis of thejoint. Non-surgical options also include injections of anti-inflammatorydrugs to inflamed tendons (such as “tennis elbow”), injection oflubricants into joints (such as hyaluronic acid into the knee), as wellas physiotherapy and bracing.

In one aspect, the tissue grafts described herein can be used to wraptendon repairs to prevent scar formation on the healing tendon. They canalso provide a protective, enclosed environment for the repair toprogress successfully. The tissue grafts can be used as an off-the-shelftendon and ligament to replace the need to purchase an allograft orperform tendon or ligament transfer.

In other aspects, the tissue grafts described herein can be used in thereinforcement of rotator cuffs. Some rotator cuff tears are large enoughthat they require a reinforcement matrix to support the repair due tolack of viable native tissue. The tissue grafts described herein can beused as a matrix to reinforce a repair. In one aspect, the tissue graftsdescribed herein can be used to repair knee cartilage. For example, thetissue grafts can be used as a barrier to hold cell culturedchondrocytes or other pro-cartilage regeneration matrix inside achondral defect. In this aspect, the tissue graft would be utilized as aflap to close the defect and hold the matrix in place.

In one aspect, the tissue grafts can be used to repair peripheralnerves. The tissue graft can be used as a wrap on nerve repairs toprevent scar formation onto the healing nerve. The tissue graft can alsoprovide a protective enclosed environment for the repair to progresssuccessfully. In other aspects, the tissue grafts can be manufacturedinto a nerve regeneration tube to guide nerve growth in a protectiveenvironment where the nerve ends cannot be re-approximated. Here, nervescan re-attach up to a certain distance if the ends are allowed to meetfreely without other soft tissue interfering. In another aspect, thetissue graft can be used to wrap nerve bundles after prostatectomyprocedures. These nerves are responsible for erectile function andpossible continence. The tissue grafts can be laid on the nerves to keepthem from scarring and possibly damaging the nerves.

In other aspects, the tissue grafts described herein can be used inother orthopedic applications such as aid in the repair of periostium;help repair ruptured/damaged bursa; help secure void filling materialduring bone repair; or in applications involving a subject's extremities(e.g., anti-adhesion barrier for small bone fixation, anti-adhesionbarrier where metal plating or hardware is used, or help repairruptured/damaged bursa).

In another aspect, the tissue grafts can be used in obstetrics andgynecological (OB/GYN) surgical procedures involving the treatment ofdiseases that may be related to the fertility of the female, pain causedby the reproductive system or cancer in the reproductive system. Theseprocedures include the removal of uterine fibroids (myomectomy), removalof ovarian cysts, tubal ligations, endometriosis treatments, removal ofsome cancerous or non-cancerous tumors, and vaginal slings. Theseprocedures may be completed through a transvaginal, abdominal orlaproscopical approach.

The tissue grafts can be used as a patch to reduce the amount of scartissue in the reproductive system after a surgical procedure. Scartissue is another form of fibrous tissue and may also contribute tofertility problems. The ability to minimize the amount of scar on theovaries, or within the fallopian tubes may help with post-operativefertility and even pain. In another aspect, the tissue grafts can beused to reline the uterine wall after severe endometriosis treatmentsand increase the patient's ability to conceive. In a further aspect, thetissue graft can be used as an anti-adhesion barrier after removal ofovarian cyst or aid in the repair of vaginal wall erosion.

In other aspects, the tissue grafts can be used in cardiac applications.Angina is severe chest pain due to ischemia (a lack of blood, thus alack of oxygen supply) of the heart muscle, generally due to obstructionor spasm of the coronary arteries (the heart's blood vessels). Coronaryartery disease, the main cause of angina, is due to atherosclerosis ofthe cardiac arteries. Various open cardiac and vascular surgeryprocedures to remove atherosclerotic clots require the repair,reconstruction and closure of the vessel, and the support of aregenerative tissue patch to close and patch the surgical defect. Heartby-pass grafts and heart defect reconstruction (as part of an open-heartsurgical procedure) also can benefit from a patch or graft to provide abuttress to soft-tissue weakness, tissue replacement if there is a lackof suitable tissue, and also the potential to reduce adhesions to theheart itself. The tissue grafts described herein can be used as a patchto support the repair of vascular and cardiac defects caused byoperations and complications such as carotid artery repair, coronaryartery bypass grafting, congenital heart disease, heart valve repair,and vascular repair (i.e. peripheral vessels).

The tissue grafts described herein can be used in general surgeryprocedures. For example, general surgical procedures include proceduresrelated to the abdominal cavity. These include the intestines, stomach,colon, liver, gallbladder, appendix, bile ducts and thyroid glands.Procedures may include hernias, polypectomy, cancer removal, surgicaltreatment of Crohn's and ulcerative colitis. These procedures may bedone open or laparoscopically. In other aspects, the tissue grafts canbe used to facilitate closure of anastomosis, an anti-adhesion barrierfor anastomosis, or an anti-adhesion barrier for hernia repair.

In other aspects, the tissue grafts can be used in ENT procedures.Tympanoplasty is performed for the reconstruction of the eardrum(tympanic membrane) and/or the small bones of the middle ear. There areseveral options for treating a perforated eardrum. If the perforation isfrom recent trauma, many ear, nose and throat specialists will elect towatch and see if it heals on its own. If this does not occur or frequentre-perforation occurs in the same area, surgery may be considered.Tympanoplasty can be performed through the ear canal or through anincision behind the ear. Here, the surgeon harvests a graft from thetissues under the skin around the ear and uses it to reconstruct theeardrum. The tissue grafts described herein can be used to prevent theadditional trauma associated with harvesting the patients' own tissueand save time in surgery. In other aspects, the tissue grafts can beused as a wound covering after adenoidectomy, a wound cover aftertonsillectomy, or facilitate repair of the Sniderian membrane.

In other aspects, the tissue grafts described herein can be used inplastic surgery procedures. Scar revision is surgery to improve orreduce the appearance of scars. It also restores function and correctsskin changes (disfigurement) caused by an injury, wound, or previoussurgery. Scar tissue forms as skin heals after an injury or surgery. Theamount of scarring may be determined by the wound size, depth, andlocation; the person's age; heredity; and skin characteristics includingskin color (pigmentation). Surgery involves excision of the scar andcareful closure of the defect. In one aspect, the tissue graftsdescribed herein can be used as a patch to aid in the healing andprevention of scars; and keloid or cancer revision/removal where carefulapproximation of soft-tissue edges is not achievable and scar tissue canresult. Additionally, the anti-inflammatory properties of the tissuegraft can enhance healing as well.

In other aspects, the tissue grafts can be used in ophthalmologicalapplications (e.g., on-lay grafts ocular surface repair) or urologicalapplications (e.g., facilitate closure of the vas deferens duringvasectomy reversal or facilitate closure of the vas deferens resultingfrom trauma).

In one aspect, the tissue grafts can be used in cranial dura repair andreplacement, in the elimination of a frenum pull, the regeneration oflost patella tissue, the repair of the Schneiderian membrane in thesinus cavity, soft tissue around dental implants, vestibuloplasty, andguided tissue regeneration.

Depending upon the application of the graft, the graft can be soakedwith a bioactive agent such as a solution composed of naturallyoccurring growth factors sourced from platelet concentrates, eitherusing autologous blood collection and separation products, or plateletconcentrates sourced from expired banked blood; bone marrow aspirate;stem cells derived from concentrated human placental cord blood stemcells, concentrated amniotic fluid stem cells or stem cells grown in abioreactor; or antibiotics. Here, one or more membrane layers of thetissue graft absorb the bioactive agent. Upon application of the wettissue graft with bioactive agent to the wound, the bioactive agent isdelivered to the wound over time.

Although the tissue grafts described herein can be applied directly tothe tissue of a subject, they can also be applied to a wound dressingthat can subsequently be applied to the subject. For example, the wounddressing can be gauze, a bandage or wrap, or any other suitable articlecapable of containing or affixing the tissue graft that can be applieddirectly to a subject.

Various modifications and variations can be made to the compounds,compositions and methods described herein. Other aspects of thecompounds, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

1. A tissue graft comprising (1) two or more layers of amnion, whereinat least one layer of amnion is cross-linked, (2) two or more layers ofchorion, wherein at least one layer of chorion is cross-linked, or (3)one or more layers of amnion and chorion, wherein at least one layer ofamnion and/or chorion is cross-linked.
 2. The tissue graft of claim 1produced by the process comprising: a. providing a first amnion having afirst surface and a second surface; and b. laminating a second amnion orchorion having a first surface and a second surface to the secondsurface of the first amnion, where the first surface of the secondamnion or chorion is laminated to the second surface of the firstamnion, and wherein the first amnion, second amnion, and/or the chorionare treated with a cross-linking agent prior to lamination.
 3. Thetissue graft of claim 1 produced by the process comprising: c. providinga first chorion having a first surface and a second surface; and d.laminating a second chorion having a first surface and a second surfaceto the second surface of the first chorion, where the first surface ofthe second chorion is laminated to the second surface of the firstchorion, and wherein the first chorion and/or second chorion are treatedwith a cross-linking agent prior to lamination.
 4. The tissue graft ofclaim 1 produced by the process comprising: a. providing a first amnionhaving a first surface and a second surface; and b. laminating a secondamnion or first chorion having a first surface and a second surface tothe second surface of the first amnion, where the first surface of thesecond amnion or first chorion is laminated to the second surface of thefirst amnion to produce a laminated article; and c. contacting thelaminated article with a cross-linking agent.
 5. The tissue graft ofclaim 1 produced by the process comprising: a. providing a first chorionhaving a first surface and a second surface; and b. laminating a secondchorion or first amnion having a first surface and a second surface tothe second surface of the first chorion, where the first surface of thesecond chorion or first amnion is laminated to the second surface of thefirst chorion to produce a laminated article, and c. contacting thelaminated article with a cross-linking agent.
 6. The graft of claim 1,wherein the amnion and the chorion are treated with the samecross-linking agent.
 7. The graft of claim 1, wherein the amnion and thechorion are treated with different cross-linking agents.
 8. The graft ofclaim 1, wherein the first surface of the amnion comprises an exposedbasement layer or an unexposed basement layer.
 9. The graft of claim 1,wherein the cross-linking agent comprises a sugar.
 10. The graft ofclaim 9, wherein the sugar comprises D-ribose, glycerose, altrose,talose, ertheose, glucose, lyxose, mannose, xylose, gulose, arabinose,idose, allose, galactose, maltose, lactose, sucrose, cellibiose,gentibiose, melibiose, turanose, trehalose, isomaltose, or anycombination thereof.
 11. The graft of claim 1, wherein the cross-linkingagent comprises a dialdehyde, a carbodiimide, or a combination thereof.12. The graft of claim 1, wherein the amnion comprises a fibroblast celllayer.
 13. The graft of claim 1, wherein the amnion and/or chorion istreated with 0.1 M to 5.0 M D-ribose for 0 to 60 minutes.
 14. The graftof claim 1, wherein the tissue graft is produced by the methodcomprising a. optionally removing substantially all of the epitheliumcells of the amnion to expose the basement membrane of the amnion toproduce a first membrane having an exposed basement membrane and asecond surface; b. treating the first membrane with a firstcross-linking agent; and c. mounting a chorion membrane having a stromallayer and a second surface to the amnion, wherein the stromal layer ofthe chorion is adjacent to the second surface of the amnion, wherein thechorion is treated with a second cross-linking agent, and wherein thefirst cross-linking agent and the second cross-linking agent are thesame or different.
 15. The graft of claim 1, wherein the graft isproduced by the method comprising: a. obtaining a placenta from asubject; b. cleaning the placenta; c. separating the chorion tissuelayer from the amnion, wherein the amnion comprises epithelium cellsadjacent to a basement membrane; d. optionally removing substantiallyall of the epithelium cells to expose the basement membrane of theamnion to produce a first membrane; e. treating the first membrane andchorion with one or more cross-linking agents; f. mounting the firstmembrane onto a surface of a drying fixture, wherein the basementmembrane of the first membrane is adjacent to the surface of the dryingfixture; g. mounting chorion on the first membrane to produce the tissuegraft; and h. dehydrating the tissue graft on the drying fixture. 16.The graft of claim 15, wherein when the epithelium cells aresubstantially removed, step (d) comprises mechanical or chemical removalof the epithelium layer to substantially remove the epithelium layer.17. The graft of claim 15, wherein the dehydrating step (h) comprisesheating the tissue graft or chemically dehydrating/freeze-drying thetissue graft.
 18. The graft of claim 15, wherein the dehydrating step(h) comprises placing the drying fixture with the layered tissue graftmounted to the fixture in a breathable bag and heating the bag in anoven or incubator chamber, wherein the oven or incubator chamber isheated at a temperature of between 35° C. and 50° C. between 30 minutesto several days.
 19. The graft of claim 15, wherein the surface of thedrying fixture has a plurality of grooves that defines the outercontours of each of the plurality of placenta.
 20. The graft of claim15, wherein the drying fixture includes a texture or design adapted toemboss such texture or design into the basement membrane duringdehydration.
 21. The graft of claim 1, wherein one or more additionalmembranes are laminated to the second surface of the amnion membrane,wherein one or more additional membranes are treated with across-linking agent.
 22. The graft of claim 21, wherein the one or moreadditional membranes comprises amnion, chorion, allograft pericardium,allograft acelluar dermis, amniotic membrane, Wharton's jelly, purifiedxenograft Type-1 collagen, biocellulose polymers or copolymers,biocompatible synthetic polymer or copolymer films, purified smallintestinal submucosa, bladder acellular matrix, cadaveric fasia, or anycombination thereof.
 23. The graft of claim 21, wherein the one or moreadditional membranes comprise an amnion membrane comprising a fibroblastcell layer, wherein one or more amnion membranes are optionally treatedwith a cross-linking agent.
 24. A tissue graft comprising amnion andchorion membrane, wherein the amnion and chorion are covalently attachedto one another via a cross-linker, and wherein the amnion comprises afibroblast cell layer.
 25. A wound dressing comprising the tissue graftof claim
 1. 26. An amnion membrane or a chorion membrane comprising atleast one cross-linker covalently attached to the membrane.
 27. A methodfor preventing or reducing scar formation on or near the spine after asurgical procedure, or for treating a dural tear, or for promoting thehealing of a wound, the method comprising applying to a subject a tissuegraft of claim 1 directly to the spinal dura of the subject or a regionnear the spine, or to the dural tear, or to the wound. 28.-53.(canceled)